Diagnostic method

ABSTRACT

A method for the identification of foetal DNA in a maternal DNA-containing sample such as a blood or vaginal sample, said method comprising (a) isolating DNA from said sample, (b) subjecting said DNA to exonucleolytic digestion by an enzyme so as to remove end regions of each DNA molecule, and (c) detecting the presence of a DNA sequence remaining in foetal DNA but absent form maternal DNA as a result of said digestion process. Once identified, the foetal DNA can be subject to diagnosis for example to detect chromosomal/DNA abnormalities, including in particular aneuploides such as foetal trisomy 21.

[0001] The present invention relates to a method for the identificationof foetal DNA, in a maternal sample such as a blood or vaginal sample.Foetal DNA identified in this way can then be used, for instance, inpre-natal diagnosis.

[0002] Chromosome disorders are among the most common genetic disease inhumans. Constitutional chromosome disorders range in incidence from morethan 50% of the lethality associated with miscarriage during the firsttrimester of pregnancy as well as around 5% of intrauterine or perinataldeaths. In addition at least 0.5% of liveborn children have aconstitutional chromosome abnormality associated with mental and/orphysical disability.

[0003] Chromosome abnormalities may be either numerical or structural.Numerical abnormalities, implying a change from the normal diploidchromosome number of 46 in somatic tissues include trisomies (one extrachromosome), monosomies (one chromosome missing) and polyploidy (wholeextra set of chromosomes). Structural rearrangements, caused bychromosome breakage followed by healing of the broken chromosome ends inaberrant positions, include so called translocations, inversions andinsertions. Structural chromosome rearrangements can occur in balancedform, in which case the genetic material usually remains the same asnormal.

[0004] Carriers of balanced structural chromosome rearrangement arephysically and mentally normal but may suffer reproductive problems withan increased risk for reduced fertility as well as an increased risk forchromosomally unbalanced offspring, leading to miscarriage, intra—orperinatal death and/or liveborn children with physical and/or mentaldisability.

[0005] The most common chromosome abnormality occurring as an entity inthe human population is trisomy 21, associated with Down Syndrome. It isgenerally accepted that around {fraction (1/650)} liveborn children hastrisomy 21 Down Syndrome characterised by more or less severepsychomotor development delay. There is no substantial difference in theincidence of trisomy 21 Down Syndrome in different countries world-wide.

[0006] The diagnosis of trisomy 21 Down Syndrome in child- and adulthoodis usually performed by chromosome analysis following in vitro cultureof blood lymphocytes. The cell culture procedure takes 2-3 days to allowaccumulation of enough cells in the metaphase stage of the cell cycle,when chromosomes are sufficiently condensed for their individualidentification by standard chromosome banding technology.

[0007] The only clearly documented clinical risk factor for having achild with regular trisomy 21 Down Syndrome concerns maternal age. Thusit is generally accepted that there is an increasing risk for having atrisomy 21 child with advancing maternal age, which in the highest agegroup of more than 45 years may be over 10% of pregnancies. Thissituation has led to intensive screening programmes of pregnant women toidentify those that are most likely to be carrying a child with trisomy21. These screening programmes include analysis of maternal bloodsamples for biochemical characteristics as well as ultrasonography ofthe foetus with the aim especially to look at the accumulation of fluidunder the skin of the neck, which is characteristically increased infoetuses with Down Syndrome and some other common aneuploidies.

[0008] In addition pregnant women over a certain age, usually 35 years,are routinely offered invasive procedures (chorionic villus samplingand/or amniocentesis) to allow foetal cell sampling for chromosomeanalysis. The most common current method for performing prenataldiagnosis of chromosome disorders concerns karyotyping, following invitro culture of amniotic fluid samples. This involves microscopyanalysis of mitotic cells, where chromosomes are sufficiently condensed.Cell cultures occupy around 1-3 weeks, and the long delay in diagnosisis recognised as being associated with much parental anxiety.

[0009] Alternative technologies for more rapid prenatal chromosomediagnosis include (1) fluorescence in situ hybridisation (FISH) ofresting (interphase) cell nuclei from uncultured amniotic fluid samples,and (2) DNA diagnosis using DNA isolated from amniotic fluid samples,amplified by for example the polymerase chain reaction (PCR) andquantity of chromosome-specific primers determined by Q-PCR techniques.

[0010] These two alternatives for prenatal diagnosis for chromosomedisorders, using amniotic fluid samples, may imply that reports can beissued rapidly, within the same or the following day after the samplehas arrived in the laboratory. So far they have been limited to therapid diagnosis of the most common chromosome abnormalities, i.e. thoseinvolving trisomies 21, 13, 18 and sex chromosome aneuploidy.

[0011] In any case, invasive methods such as amniocentesis and chorionicvillus sampling, as well as being uncomfortable for the mother, areassociated with an increased risk of miscarriage of around 1-1.5%. Thereis a need therefore to provide a more efficient way of carrying outpre-natal diagnosis without resorting to such invasive sampling methods.

[0012] Samples obtained using less invasive methods from the pregnantmother will commonly contain a vast majority of maternal cells with arelatively small number of foetal cells, in the order of 1 per 10,000 to1 per 10 million. Current methods for foetal cell isolation include theuse of antibodies, gradient fractionation, preferential maternal celllysis, magnetic activated cell sorting (MACS), ferrofluid suspensionwith magnet, micromanipulation of individual cells, charged flowseparation and fluorescence activated cell sorting (FACS). However,maternal cells still tend to dominate any foetal cells recovered. Inaddition, many of these techniques are time consuming and labourintensive.

[0013] It has more recently been recognised that maternal blood samples,in particular plasma or serum contain relatively large amounts of foetalDNA (Lo et al., Lancet 1997, 350, 485-487, WO 98/39474). It has furtherbeen demonstrated that this foetal DNA, following in vitro amplificationby techniques such as PCR may be identified with respect tofoetus-specific sequences. This technology has been applied on severaloccasions for diagnosis of foetal sex by Y-chromosome specific primers,and other conditions such as Haemoglobinopathies, where prenataldiagnosis is based on specific mutations in foetal DNA.

[0014] In addition it has been shown that foetal chromosomeabnormalities such as trisomy 21 is associated with an increase infoetal DNA in maternal serum/plasma in comparison to the normalsituation using foetal-specific primers as exemplified above.Furthermore complications in pregnancy such as pre-eclampsia and pretermlabour and the post partum development of autoimmune disease, may alsobe characterised by increased fetomaternal transfusion, leading tohigher levels of fetal cells in maternal blood (reviews in Pertl andBianchi Semin Perinatol 23, 5, 393-402, 1999; Bianchi Eur J ObstetGynecol Reprod Biol 92, 1, 103-8, 2000).

[0015] It has been concluded that the amount of foetal DNA, ascertainedby foetal-specific DNA primers, (such as Y-chromosome specific primersin male foetuses) may be used as a screening method for the detection ofpregnancies, where there is an increased risk for foetal chromosomeaneuploidy. Most importantly however, it has not been possible todiagnose foetal aneuploidy per se by said methods. Thus these methodsare not sensitive enough to measure the difference between foetal DNA,to determine whether the foetus is normal or aneuploid. Examples of thesorts of results these tests give are illustrated in FIG. 1 hereinafter.In FIG. 1a, the foetus is normal and there are two peaks correspondingto the maternal alleles. One of these (M2) is larger than the other (M1)and there is an additional amount of DNA originating from the foetal DNA(F). The size of this is the same as the peak originating from thefoetal allele of paternal origin (F-Pat).

[0016] In FIG. 1b, the foetus has trisomy 21 with one copy of F on eachof the maternal alleles M1 and M2, plus one copy of paternal origin(F-Pat). This will reduce the difference in fluorescence between thealleles M1 and M2 in comparison to the normal situation illustrated in(a). In FIG. 1c, the foetus has trisomy 21 with two copies of F onmaternal allele M2, plus one copy of paternal origin (F-Pat). Thisincreases the difference in fluorescence between the alleles M1 and M2in comparison to the normal situation illustrated in FIG. 1a. Finally inFIG. 1d, the foetus has trisomy 21 with 2 copies of paternal originF-Pat. This makes no difference to the fluorescence from the maternalalleles M1 and M2, but the fluorescence of the paternal allele isdoubled. Such results are very difficult to interpret, and there is toour knowledge no documented case where prenatal diagnosis of trisomy 21(or any other aneuploidy) has been diagnosed in this way, using amaternal blood or vaginal sample.

[0017] There is therefore an urgent need for new methods to identifyfoetal DNA in a maternal DNA-containing sample such as a blood orvaginal sample, comprising a novel way of non-invasive prenataldiagnosis.

[0018] It is well known that the telomeres, constituting repeated DNAsequences that cap the ends of chromosomes, vary such that young peoplehave a higher number of the repeats than older people. It is thoughtthat DNA replication is not taking place at the very ends of thetelomere repeats. This means that, at each cell division, the telomeresbecome shorter than before. It is also thought that this shorteningeventually leads to cell death.

[0019] Telomeres of all human chromosomes contain the same DNA corerepeat TTAGGG. The variation in telomere length with age of theindividual is a general phenomenon observed on all the chromosomes.Depending upon the age of the individual, variation in repeat length oftelomeres is estimated to be in the order of 4-200 Kb of DNA.

[0020] Chromosome-specific telomere lengths can be measured usingspecial software and microscopy image analysis of chromosomes hybridisedwith telomeric probes or commercially available peptide nucleic acidprobes (PNA, DAKO Ltd). These investigations indicate that there may besome variation between individual cell nuclei in the telomere content ofindividual chromosomes. Nevertheless, as already mentioned, there is asubstantial decrease in telomere length with the age of the subject.

[0021] On this basis, the telomere length of individual chromosomes infoetal cells should be longer than in the newborn child, and longerstill than in the adult. It is implicit therefore that foetal cells havelonger telomeres, i.e. a higher copy number per chromosomes of thetelomere DNA repeats, than cells from the mother (Butler et al., CancerGenetics and Cytogenetics 105, 138-144, 1998; Kreji and Koch, Chromosoma107, 198-203, 1998).

[0022] The applicants have found that this characteristic can be used asa basis for differentiation between maternal and foetal DNA, inparticular DNA, present in a maternal DNA-containing sample such as ablood (including plasma or serum) or vaginal sample.

[0023] Thus according to the present invention there is provided amethod for the identification of foetal DNA in a maternal DNA-containingsample, said method comprising (a) isolating DNA from said sample, (b)subjecting said DNA to exonucleolytic digestion by an enzyme so as toremove end regions of said DNA, and (c) detecting the presence of a DNAsequence remaining in foetal DNA but absent from maternal DNA as aresult of said digestion process.

[0024] Steps (a) and (b) may be conducted in any order. For example, DNAis first isolated from the cells and then subject to enzymatic digestionin accordance with step (b). However, alternatively, the digestion maybe effected directly in cells, and digested DNA isolated for example byPCR amplification therefrom for analysis in step (c).

[0025] Suitably the maternal DNA-containing sample is a blood or vaginalsample. Preferably, the method is carried out using a maternal bloodsample. The expression “blood sample” as used herein encompasses wholeblood, or serum or plasma derived therefrom.

[0026] In a particularly preferred embodiment, cells are first separatedfrom maternal plasma and DNA extracted from these as step (a), or ifappropriate during or after step (b), of the process. Examples of suchDNA amplification, which is advantageous when only a small amount ofmaterial is available, are described for instance in Findlay et al MolPathol 51 (3), 164-167, 1998, Klein et al Proc Natl Acad Sci USA 96 (8),4494-4499, 1999.

[0027] However there may be cases were the invention is convenientlyemployed in the analysis of other prenatal sample types such as amnioticfluid. Generally amniotic fluid samples contain relatively littlematernal DNA and identification of foetal DNA is not difficult. On someoccasions however, the samples are contaminated with maternal blood, andgive complicated fluorescence patterns when analysed for example usingthe Q-PCR method. Isolation of foetal DNA from such samples using amethod of the invention as a preliminary step might be useful.

[0028] DNA may be isolated from the sample using conventional methods.Preferably techniques are employed which result in the isolation of longDNA fragments. An example of such a method is the use of agarose plugsas described by Heiskanen et al., Biotechniques 17, 5, 928-929; 9320933,1994.

[0029] In one embodiment, the DNA from step (a) is cut using arestriction enzyme into fragments prior to step (b). Thus theexonucleolytic digestion removes terminal regions of the DNA fragments(See FIG. 2 hereinafter). [However, this may not always be necessary, inparticular where cells isolated from plasma are themselves directlysubject to exonucleic digestion, and the resultant DNA amplified by PCRprior to analysis.]

[0030] In a particularly preferred embodiment, cut ends of the DNAfragments are protected prior to the exonucleolytic digestion so thatthey are not susceptible to digestion by exonucleolytic enzymes such asBal31. This will mean that the digestion of fragments derived from theterminal regions of the DNA will be unidirectional, from the telomereregion inwards. The proximal end will be protected from digestion, aswill fragments derived from internal regions of the DNA. As a result,digestion will take place only in the telomere region (See FIG. 3hereinafter).

[0031] Suitably, the restriction enzyme used to form the DNA fragmentsis one which cuts so as to provide binding sites for a protectingmoiety, such as an adaptor. Protection can then be effected by ligationof a suitable adaptor. A suitable adaptor may be 2′-O-methylribonucleotide DNA (Mukai et al. Nucleic Acid Research Symposium Series19, 1998) or complementary oligonucleotide containing phosphorothiatelinks.

[0032] Suitable DNA sequences remaining in foetal DNA which may bedetected in step (c) is a telomere sequence or a chromosome markerlocated proximal to the telomere region, and is most preferably asubtelomeric sequence, in particular one which is unique to the specificchromosome.

[0033] Detection in step (c) may be effected by any of the knowntechniques. In a preferred embodiment however, foetal DNA is isolatedand purified using magnetic separation techniques. A particular methodby which this can be achieved involves the use of biotinylated primersas probes specific for telomeres. Hybridisation of primers will meanthat the foetal DNA will have a biotin label which can be separatedmagnetically using streptavidin coated paramagnetic particles (PMPs) orbeads (see for example FIG. 4).

[0034] In context of the methods of the invention, it is frequently thecase that primers can be used to function as probes. It will be clear tothe skilled person where this applies. Thus the term “primer” as usedherein should be understood as referring to sequences which may haveprobe function, as well as sequences which are used as conventionalprimers for amplification reactions.

[0035] Once isolated in this way, the DNA fragments may be analysed. Ifnecessary, they may first be subjected to amplification. Pure amplifiedfoetal DNA obtained in this way may have a wide application innon-invasive prenatal genetic analysis and diagnosis. It may beparticularly preferred to carry out quantitative analysis for example,using a fluorescence Q-PCR method for instance by Applied Biosystems DNAsequencers and the Genescan software, as well a Pyrosequencing and othersuch methods including Microarrays. Many of these methods are nowbecoming fully automated.

[0036] In effect, the invention uses differences in the number oftelomere repeats in foetal and maternal DNA as a basis foridentification of foetal DNA in maternal DNA-containing samples. Duringstep (b), DNA, preferably in the form of fragments and most preferablylong fragments, is digested, preferably unidirectionally followingprotection of cut ends, from the telomeric end regions of the fragmentsinwards.

[0037] Telomere regions of all chromosomes present in the sample aredigested first during this process. Exonucleolytic digestion may becarried out for a period of time sufficient to eliminate at least allmaternal telomeric DNA sequences.

[0038] If digestion is halted at this point, some foetal DNA fragmentswill retain some telomeric DNA (FIG. 3a). This DNA is then detectableusing for example, a labelled primer specific for the primer telomereDNA which will therefore hybridise only to foetal DNA.

[0039] Digestion may however be continued such that subtelomeric DNAsequences are eliminated from all fragments of maternal DNA (FIG. 3b).In this instance, because digestion of the longer foetal telomeres takeslonger, some chromosome-specific DNA will remain at the correspondingsite in fragments of foetal DNA and so will be detectable for exampleusing a primer for that DNA which therefore acts as a marker. The primerwould not hybridise to maternal DNA under these circumstances.

[0040] Preferably, primers used in the detection are labelled withvisible labels such as fluorescent labels.

[0041] Where the sequence detected in step (c) is a chromosome marker,it may be preferable that it is a sub-telomeric polymorphic chromosomemarker, as this gives rise to the possibility that the marker can itselfbe readily useful in pre-natal diagnosis.

[0042] In any event, the identification of foetal DNA can be used as apreliminary step to pre-natal diagnosis of the identified DNA.

[0043] In a particularly alternative embodiment, DNA present in thesample after digestion is amplified with a first labelled DNA primerspecific for the said DNA sequence, such as the telomeric and/orsubtelomeric sequence. Most preferably the first primer is labelled witha visible label in particular a fluorescent label, and fluorescence fromthe amplified sample is detected.

[0044] Suitable enzymes for conducting exonucleotylic digestion includeBal31. It may be preferable to use enzymes which digest specific regionsof the DNA only, in order to ensure a more controllable digestionprocess. In particular, digestion is effected in a three step process,in which, in a first step, 3′ extension DNA is removed, in a secondstep, 3′-5′ ss regions are excised and in a third step, ss regions aredigested. Suitable enzymes for effecting the first and third stepsinclude Mungbean nuclease, and for the second step, suitable enzymesinclude Exonuclease III.

[0045] The conditions, such as enzyme concentrations, buffer systems,temperature and time of incubation, required in order to providereliable digestion to allow differentiation between maternal and foetalDNA, require careful selection and depend upon factors such as theparticular enzyme being used.

[0046] As a result of the variation in the numbers of telomeric DNAsequence repeats between different DNA ends, it is desirable first to“calibrate” the enzyme system, preferably in a chromosome specificmanner. Calibration of this type may be effected by analysing theresults of exonucleolytic telomeric digestion of the isolated DNA undervarious conditions.

[0047] Another means of calibrating particular enzyme systems is toobtain base-line information on the telomeric length of each individualchromosome end in maternal and foetal tissue samples. This may be doneusing fluorescence in situ hybridisation (FISH) of telomeric DNAsequences at the metaphase stage of the cell cycle. At this stage theindividual telomeres at each chromosome end may be highlighted usingFISH with telomere-specific probes in combination with subtelomeric DNAprobes. Measurements of telomeric sequences may be performed byMicroscopy Image Analysis, using for example a Comparative GenomicHybridisation (CGH) software programme.

[0048] Blood samples from foetuses subject to cordocentesis and cordblood samples, obtained at delivery, can also be utilised, with a viewto obtaining additional base-line information on the normal variation inlength of telomeric DNA sequences for each chromosome arm in maternaland foetal tissue samples.

[0049] The conditions of enzyme concentrations and exposure timesrequired to eliminate a subtelomeric marker from maternal DNA can bedetermined in a similar manner. In this instance, amplification forexample using the polymerase chain reaction (PCR) of the subtelomericmarkers of the DNA product may be effected in order to distinguishbetween constitutional maternal DNA alleles and foetal DNA alleles. Thisexperimentation will provide base-line information on the rate ofremoval of subtelomeric DNA sequences by the respective enzymaticdigestion conditions applied.

[0050] Preferred subtelomeric markers include, for instance a smalltandem repeat (STR) or a microsatellite. For this purpose, they may ormay not be polymorphic markers as they are only used for evaluation oftheir rate of disappearance by the exonucleolytic digestion and fordifferentiation between constitutional maternal and foetal DNA.

[0051] Polymorphic diagnostic markers may be preferred however if theanalysis is subsequently used for prenatal diagnosis. Examples ofchromosome 21—specific polymorphic tetranucleotide repeat markers areD21S11, D21S1412, D21S1411 and D21S1414. IFNAR, which is a dinucleotiderepeat also shows a simple amplification pattern, in contrast to mostother smaller repeat markers, which may be associated with stutterbands. Other examples of polymophic markers include single nucleotidepolymorphisms (SNPs), which occur with very high frequency in the humangenome, and where an increasing number are currently being identified.

[0052] In this case, the concentration of the foetal marker maysubsequently be determined, for example using quantitative PCR methodssuch as TAQMAN™. This information, can be useful in pre-natal diagnosisof foetal conditions where only the amount of certain DNA sequences aredifferent between the foetus and the mother. A reliable quantificationof amount of chromosome-unique foetal DNA is required for thenon-invasive prenatal diagnosis of common foetal conditions, includinganeuploidies, such as Trisomy 21 Down syndrome, using a maternal bloodsample.

[0053] In a preferred option the subtelomeric markers in question shouldbe located as distal as possible among the chromosome-unique DNAsequences within each chromosome arm. These markers will be preselectedfor each chromosome end separately with respect to locations and degreeof polymorphisms in the population studied.

[0054] Suitably the DNA markers selected are strategically localised insubtelomeric chromosome positions, containing chromosome-specific uniqueDNA. These locations are preferred for two main reasons.

[0055] Firstly, markers, localised near to the telomeres but yetcontaining unique chromosome specific DNA, will facilitate the digestionprocedure, as only limited exonucleolytic digestion will be required incomparison to that needed to selectively eliminate maternal, moreproximal, interstitial, DNA. This will be particularly useful whenunidirectional digestion is undertaken as described above.

[0056] Secondly, the use of polymorphic subtelomeric Chromosome/DNAmarkers will allow quantification of not only numerical chromosomeaberrations (such as trisomies) but also unbalanced structuralchromosome rearrangements such as unbalanced translocations. Anunbalanced translocation may be identified as a duplication of dosagefor one subtelomeric marker in combination with a deletion of dosage foranother subtelomeric marker, dependent on which chromosomes are involvedin the translocation. In addition, other relatively common chromosomeaberrations should be identifiable this way. These include extra markerchromosomes such as foetal iso 12p, or iso 18p, both associated withsevere foetal malformations, which would give rise to extra doubledosages in relation to the normal situation.

[0057] Special exonucleolytic procedures may be required for thediagnosis of extra chromosome markers such as 15 inv dup, as thisinvolves two extra doses of 15p telomeric DNA sequences. Suchduplications of telomeric sequences alone are harmless with respect tofoetal development, while markers that also contain chromosome15-specific DNA, located in the proximal part of the q arm, areassociated with foetal psychomotor development delay, which may besevere.

[0058] By appropriate selection of subtelomeric markers used in theprenatal foetal diagnosis, the majority of chromosome abnormalities,leading to foetal development disturbance may be determined using thismethod. The only chromosome abnormalities that may not be detectableusing the method, applying subtelomeric DNA markers alone, are moreproximal (interstitial) deletions and duplications that are unexpectedand therefore their exact locations cannot be determined. Specialmodifications of the method will also be required to diagnose specifieddeletions and duplications, expected because either parent is a carrier.

[0059] Once identified using the method of the invention, foetal DNA,may be subject to pre-natal diagnosis, for example to determine thepresence of chromosome aberrations such as aneuploidy, Down syndrome,Edward Syndrome or Klinefelter Syndrome or other information such as thesex of the foetus.

[0060] Suitably, DNA is separated prior to analysis, for example using amagnetic separation method. In particular, the DNA is amplified using alabelled primer such as a specific biotinylated primer. If say atelomeric primer is used at this point, only telomere-containing, i.e.foetal DNA fragments will be measured and labelled, assuming all thecorresponding maternal DNA has been eliminated during the digestion.

[0061] Said DNA fragments may then be captured using for examplestreptavidin coated para-magnetic particles or beads. After separationof the magnetic particles from the supernatant, the target DNA can bereleased by elution of the particles (FIG. 4).

[0062] Once separated, said DNA may be amplified with a labelled-primerwhich is specific for a particular chromosome or diagnostic region ofDNA under conditions in which the primer amplifies DNA within thesample. Detection of this primer in DNA identified as being of foetalorigin will therefore provide information about the foetus. Dependingupon the particular diagnostic purpose, it may be useful if the secondprimer is specific for chromosome 18, 21 or 13, or the X or Ychromosomes. Abnormalities in chromosome number (aneuploidies) of thesechromosomes are the most common.

[0063] In a particularly preferred embodiment of the invention, DNA isisolated from a maternal blood sample, for example, from cells inmaternal plasma, fragmented with a restriction enzyme, and then ligatedto an adaptor. The modified fragments are then incubated with anexonucleolytic enzyme such as Bal31, for a period of time which issufficient to entirely digest maternal telomeres, but leave some foetaltelomeric DNA. Further purification of teleomere-containing (foetal) DNAfragments may be achieved by magnetic separation using biotinylatedtelomeric probes or telomeric primers used as probes and streptavidinparamagnetic particles (PMPs) (FIG. 4).

[0064] Isolated telomere-containing (foetal) DNA fragments are thenamplified by a method such as PCR, using chromosome-specific primers.Analysis is thereafter performed using a fluorescent method such asQ-PCR utilising, for instance, a DNA sequencer and Genescan software(Applied Biosystems), real time PCR or pyrosequencing.

[0065] In addition, the information obtainable using the method of theinvention, in particular relating the amount of concentration of foetalDNA in the maternal sample, may be useful in prenatalscreening/diagnosis as well as in diagnosis of a range of maternalconditions. These include complications in pregnancy such aspre-eclampsia, in predicting the risk of pre-term labour, and in thelater development of autoimmune disease.

[0066] According to a further aspect of the invention, there is provideda kit for identifying foetal DNA in a maternal DNA-containing sample,said kit comprising means for isolating DNA from a DNA-containingsample, an exonucleolytic enzyme capable of digesting terminal regionsof DNA, and a labelled primer for detecting a specific DNA sequencefound in a terminal region of DNA.

[0067] The kit may contain one or more further reagents or commoditieswhich are required for effecting the method as described above. Inparticular, the kit may further comprise a restriction enzyme which cutsDNA so as to produce a binding site on the DNA for an adaptor, togetherwith the adaptor suitable for protecting the cut ends of DNA fragmentsfrom enzymatic digestion.

[0068] Depending upon the way the assay is operated, biotinylatedprimers and particularly biotinylated telomeric primers as well asstreptavidin paramagnetic particles may also be included.

[0069] Other potential kit components include one or more additionallabelled primers, such as a different fluorescently labelled primer,which is diagnostic of a chromosome condition.

[0070] The invention will now be particularly described by way ofexample with reference to the accompanying diagrammatic drawings inwhich:

[0071]FIG. 1 shows a comparative example of Genescan analysis of DNAextracted from maternal blood (a) where the foetus is normal and (b),(c) and (d) where the foetus has trisomy 21, as carried out using priorart method.

[0072]FIG. 2 illustrates diagrammatically the exonucleolytic digestionof DNA fragments such as Bal31, where fragments are digested at bothends: although there are fewer telomeric sequences in the maternalsequences (M) in comparison to the foetal sequence (F) and so allowfoetal DNA to be identified, differentiation between M and F fluorescentmarker signals, if positioned as shown, would not be possible;

[0073]FIG. 3 illustrates diagrammatically the unidirectionalexonucleolytic digestion of DNA fragments using an adaptor in accordancewith a preferred embodiment of the invention; such that in an optimaltime course, the enzyme will eliminate all M telomeric sequences (FIG.3a) and also the M fluorescent marker signal (FIG. 3b). Such a systemtwill allow differentiation of M and F fragments with selective retentionof F fluorescent signal allowing enumeration of the F chromosomesconcerned following DNA amplification e.g. by Q-PCR; and

[0074]FIG. 4 illustrates diagrammatically the magnetic purification offoetal DNA using a biotinylated telomere probe.

EXAMPLE 1

[0075] Step 1

[0076] DNA Extraction

[0077] 5 mls of maternal blood was taken into each of two tubes, one ofwhich contained EDTA. Blood samples were centrifuged at 3000 g andplasma and serum were carefully removed, into clean tubes, from theEDTA-containing and plain tubes, respectively. The plasma and serumsamples were centrifuged again at 3000 g and the supernatants weretransferred into clean tubes.

[0078] DNA was extracted from the plasma and serum samples using theQIAamp Blood Kit from Qiagen, according to the manufacturers'recommendations.

[0079] Step 2)

[0080] Restriction Enzyme Digest to Provide Adaptor Attachment Sites

[0081] The extracted DNA is digested with enzymes such as Notl usingstandard restriction enzyme digestion protocols.

[0082] Step 3)

[0083] Ligation of DNA Template and Adaptor to Protect the Proximal Endsof DNA Fragments from Digestion by Exonucleolytic Enzyme (Such as Bal31)

[0084] This approach concerns a unidirectional DNA deletion with Bal 31nuclease (Mukai S., Shibahara S., Morisawa H. Nucleic Acids ResearchSymposium Series No. 19, 1998). The principle is based on the fact that7 bp of 2′-O-methyl ribonucleotide-DNA chimeric adapter forms greaterstability and also due to steric hindrance prevents the action ofnuclease attack. Therefore, ligation to Notl sites on the DNA fragmentswill allow digestion from one end only.

[0085] DNA and adaptor are incubated overnight at 15° C. with Ligase andbuffer.

[0086] Step 4)

[0087] Digestion of Ligated DNA Templates and Adaptor withExonucleolytic Enzyme Such as Bal 31

[0088] 1 unit of Bal 31 per microgram of DNA was used to yield a uniformladder of deletions (>1 kb) over a 20 minute period at 30° C. Thereaction was stopped by adding EDTA to a final concentration of 50 mM.(FIG. 3). DNA is ethanol precipitated, to remove the high NaClconcentration from the Bal 31 reaction. Variation in conc. and/or timingof exposure is optimised for each chromosome enumeration. In theoptimised situation all maternal telomeric sequences will be digested,while some of foetal telomeric DNA sequences will remain (FIG. 3a). Thisallows differentiation between maternal and foetal DNA fragments per se.

[0089] Extended digestion will eliminate the DNA sequences correspondingto the chromosome-specific primer site (FIG. 3b), which will allowpositive identification of foetal DNA and enumeration of the respectivechromosome, following Q-PCR.

[0090] Step 5)

[0091] Magnetic Purification of Foetal DNA Using Biotinylated TelomereProbe

[0092] DNA fragments are hybridised in solution with a Biotinylatedtelomere DNA primer by heating the DNA at 65° C. for 10 mins, adding thebiotinylated primer and leaving the solution to cool down at roomtemperature. Thereafter streptavidin paramagnetic particles (SA-PMP) arewashed with 0.5×SSC. The annealed DNA fragments and biotinylatedtelomeric primer are added and incubated at room temperature for 10mins. SA-PMPs bound to the biotinylated primer are captured using amagnetic stand, and the supernatant is carefully removed. The particlesare washed 4 times with 0.1×SSC. Telomere positive (foetal) fragmentsare eluted by resuspending the final SA-PMP particles in deionisedwater.

[0093] Step 6)

[0094] Q-PCR Using Chromosome Specific Polymorphic Markers

[0095] Quantitative PCR was carried out using standard methodology asdescribed in our publication Verma et al., The Lancet 352, 912, 1998.

EXAMPLE 2

[0096] A protocol for conducting analysis using the method of theinvention is illustrated in Scheme 1 hereinafter.

[0097] Step 1

[0098] Preparation of Blood Samples on Percoll Gradient

[0099] Blood samples are prepared on Percoll gradients and the plasmaremoved. Plasma cells are recovered by centrifugation using amodification of the standard protocol for discontinuous Percoll gradient(see e.g. Van Wijke et al. Clinical Chemistry 46, 5, 729-731, 2000).

[0100] In particular, 3 mls each of 40%, 45% and 50% Percoll solution(Amersham Pharmacia) in PBS, for each tube are prepared. 3 mls of EDTAblood are mixed with 3 mls of PBS in a 15 ml Polystyrol tube (Sarstedt,Roher 15 ml tubes cat No. 62.553.042). 3 ml of 40% Percoll is introducedas an underlayer using a canula (B. Braun, Filter Straw cat No. 415020)attached to a 5 ml syringe. The tip of the canula is placed at thebottom of the tube going through the blood and the Percoll solutionejected slowly. Using the same canula, the underlayering process is thenrepeated twice with 45% and 50% Percoll respectively. In each case, thetip of the canula is placed at the bottom of the tube and the ejectionis made carefully so as not to disturb the gradients.

[0101] In general 3-4 tubes will be prepared in this way for each sampleunder test. The prepared tubes are then centrifuged (without using thecentrifuge brakes) at 500 g for 30 minutes at 18-20° C.

[0102] After this, the tubes are removed and the plasma (top) layertaken off and put in a clean tube. Plasma from different tubes from thesame sample may be added together to ensure that there is about 6 mls offluid in each tube. This is then centrifuged again at 500 g for 20minutes, this time with the brakes on. The resultant cell pellet is thenwashed in 6 ml of PBS a couple of times.

[0103] Step 2.

[0104] Extraction of High Molecular Weight DNA

[0105] Plasma cells¹ are resuspended in PBVS at 2×10⁵ cells per 50 μl².Equal amounts of agarose solution (1.9% NuSieve GTG) are added to cellsuspension-containing 1×10⁵ cells. 50 μl are dispensed into molds andallowed to set. Agarose plugs are then treated in: 0.5M EDTA, pH 8.0, 1%N-Laurylsarcrosine, 2 mg/ml Proteinase K for 24-72 hours at 50° C.(although O/N incubation may be sufficient for our downstreamapplications). This treatment digests cell membrane allowing penetrationof enzymes in later stages. The agarose plugs are then washed in waterand incubated at 50° C. in PMSF (a protease inhibitor) to fullyinactivate any remaining Proteinase K that will effect enzymatictreatment. Agarose plugs are then washed and stored at 4° C.

[0106] Note:

[0107] 1. 3 ml blood contains ˜2×10³ plasma cells

[0108] 2. 1×10⁵ cells=1 μl DNA

[0109] Plasma DNA is extracted with phenol:chloroform (1:1), ethanolprecipitated, washed twice in 70% ethanol and carefully resuspended inwater.

[0110] Step 3

[0111] Ligation of Nuclease Resistant Adapters

[0112] This procedure may be omitted if the ligation of adapters isunnecessary, as this is the only step that is affected by the DNA beingin the agarose plugs. Both Bal31 digestion and the PCR reaction will beunaffected by the agarose plugs. The protocol allows the agarose to bedigested into alcohol soluble oligosaccharides, this makes the DNA moreaccessible without damaging or shearing the DNA. The agarose plugs aremelted and an equal volume of 50× buffer is added plus 1U β-Agaraseincubated at 45° C. for 60 minutes. The remaining oligosaccharides andβ-Agarase do not interfere with subsequent enzymatic reactions.

[0113] For the ligation of adapters the DNA will have to be cut with arare cutter such as Asc I or Not I which cut within the human genome ˜every 670 kb and 310 kb respectively. This will produce compatible endsfor the adapter to ligate to, restriction sites between the telomere andmarker will have to be checked to ensure that these enzymes do not cutwithin this region. Equilibrate the agarose plug in 100 μl of 1×restriction buffer with 10U of enzyme, incubate for 2 hours and thenrinse with water.

[0114] Complementary oligonucleotides containing phosphorothioate linkswill be annealed to produce adapters resistant to nuclease digestion.They will also contain cohesive ends complementary to the restrictionendonuclease used to cleave the DNA samples. These adapters will beligated by the addition of 1× ligation buffer, 5U Ligase and incubatedat 37° C. for 2 hours.

[0115] Step 4.

[0116] Bal31 Nuclease Digestion of Telomere Ends

[0117] Agarose plugs containing high molecular weight DNA areequilibrated for 30 minutes in 1 ml of 1× Bal31 buffer with 50 U ofenzyme (NEBL) on ice for 30 minutes. Digestion is effected by placingthe reaction at 30° C., removing plugs at each time point i.e. 0, 30,60, 90 and 120 minutes and placing in 1 ml ice-cold TE. Bal31 is removedby rinsing plugs in water for 30 minutes on ice. Bal31 should becompletely inactivated during the heat denaturation step of the PCR.Time points are taken initially to establish the length of digestionneeded to remove the required length of DNA to allow discrimination ofthe fetal DNA with the longer telomeres.

[0118] Step 5

[0119] Amplification of Polymorphic Markers by QF-PCR

[0120] Polymorphic markers are amplified by QF-PCR using standardtechnology (see e.g. Verma et al., Lancet, 352, 9-12, 1998; Pertl etal., Amer. J. Obstet. Gynecol. 177, 4, 899-906).

[0121] The X22 marker (˜300 kb from telomere) is a particular markerwhich may be amplified in this way, but other telomeric polymorphicmarkers may be selected depending upon the particular aims of thediagnosis. For example, two highly polymorphic markers on chr14 D14S1419and D14S1420 are located at ˜210 kb and 95 kb from the telomere.

EXAMPLE 3 Non-Invasive Prenatal Diagnosis of Foetal Trisomy 21 DownSyndrome Using the Methods of the Invention EXAMPLE 3A

[0122] 1. Pregnancy and Blood Sample

[0123] 12 ml of blood was drawn into 2 edetic acid (EDTA) tubes byvenipuncture from a pregnant woman at 12 weeks gestational age,following written informed consent with Ethical Approval from the LocalEthical Committee.

[0124] 2. Isolation of Cells from the Nuclear Compartment and Plasma

[0125] Both nucleated cells from the nuclear compartment and from plasmawere isolated from the maternal blood using a Triple Density Gradientaccording to the protocol as described (Ganshirt et al., DiagnosticCytogenetics, Springer Lab Manual, 1999 R. -D. Wagner, Fetal Cells inMaternal blood, pp 401-415) with slight modifications.

[0126] 12 ml of the EDTA blood is added to 12 ml of Phosphate BufferSolution (PBS) and mixed by inverting the tube. 6 ml of the blood/PBSmixture is pippetted into four 15 ml polystyrol tubes. Three layers ofPercoll^(R) (Amersham Pharmacia) are underlayered, using a long and thincanula attached to a syringe. Initially 3 ml of 40% Percoll isunderlayered, followed by 3 ml of 45% and 3 ml of 50% Percoll. Thesuspension is then centrifuged at 500 g for 30 min. The plasma layer andthe lymphocyte layer are removed and transferred to clean tubes andagain centrifuged at 500 g for 10 min. The cell pellet is washed in PBSand transferred to microtubes containing 5 μl proteinase K solution (400mg/l proteinase K, 20 mmol/l dithiothreitol, 1.7 μmol/l sodium dodecylsulfate, 10 mmol/l Trisbuffer, 50 mmol/l potassium chloride).

[0127] 3. Extraction of High Molecular Weight DNA

[0128] Plasma cells are resuspended in PBVS at 2×10 cells per 50 μl.Equal amounts of agarose solution (1.9% NuSieve GTG) are added to thecell suspension, containing 1×10⁵ cells. 50 μl are dispensed into moldsand allowed to set. Agarose plugs are then treated in: 0.5M EDTA, pH8.0, 1% N-Laurylsarcrosine, 2 mg/ml Proteinase K for 24-72 hours at 50°C. The agarose plugs are then washed in water and incubated at 50° C. inPMSF (a protease inhibitor) to fully inactivate any remaining ProteinaseK that will effect enzymatic treatment.

[0129] 4. Ligation of Nuclease Resistant Adapters

[0130] The agarose plugs are melted and an equal volume of 50× buffer isadded plus 1U β-Agarase and incubated at 45° C. for 60 minutes. Theagarose plug is equilibrated in 100 μl of 1× restriction buffer with 10Uof enzyme, incubated for 2 hours and then rinsed with water.

[0131] These adapters are then ligated by the addition of 1× ligationbuffer, 5U Ligase and incubated at 37° C. for 2 hours.

[0132] 5. Bal31 Nuclease Digestion of Telomere Ends

[0133] Agarose plugs containing high molecular weight DNA areequilibrated for 30 minutes in 1 ml of 1× Bal31 buffer with 5 U ofenzyme (NEBL) on ice for 30 minutes. Digestion is effected by placingthe reaction at 30° C., removing plugs at each time point i.e. 0, 30,60, 90 and 120 minutes and placing in 1 ml ice-cold TE. Bal31 is removedby rinsing plugs in water for 30 minutes on ice.

[0134] 6. Amplification of Polymorphic Markers by QF-PCR

[0135] A multiplex fluorescence PCR assay is applied by routinetechnology (see e.g. Pertl et al 1997 Am. J. Obstet. Gynecol. 177,899-906) using primers for D21S1411 and D21S1446. Each of the forwardprimers is labelled (5′ end) with a fluorescent dye to enablevisualisation and analysis of the PCR product. After the initialdenaturation, 24 cycles of 95° C. for 48 sec, 60° C. for 48 sec, and 72°C. for 1 min is performed, followed by an extension of 72° C. for 15min.

[0136] 7. Analysis

[0137] The allelic fragments are resolved on a 6% denaturingpolyacrylamide gel, and analysed on an Applied Biosystems (Warrington)373 DNA sequencer running Genescan 672 software. Amplification productsare sized, and their fluorescence intensities calculated, based on theareas of the peaks seen on the electropherograms. Analysis was performedon (a) DNA from lymphocytes of the maternal blood sample directly, (b)DNA from same type of cells after enzymatic digestion, and (c) DNA fromcells in the plasma component of the maternal sample (expected tocontain an enrichment of fetal cells) after enzymatic digestion.

[0138] 8. Results

[0139] (a) The electropherogram obtained from PCR products of DNA of thelymphocytes of the maternal blood sample shows the occurrence of twoequal sized peaks for each of the two primers D21S1411 and D21S1446.This result is the expected from a normal female.

[0140] (b) The electropherogram obtained from PCR products of DNA of thelymphocytes from the maternal blood sample, following enzymaticexonucleolytic digestion with 2 Units of Bal31 for 20 min, shows absenceof signals for the most distal marker D21S1446, while two equal sizedsignals are seen at positions for the proximal marker D21S1411.

[0141] This indicates that these conditions for enzymatic digestion areoptimal for our purposes of differentiating between maternal and fetalDNA.

[0142] (c) The electropherogram obtained from the DNA of cells inmaternal plasma, following enzymatic digestion under same conditions asin (b) shows unclear signals for the proximal marker D21S1411 but threeclear signals for the distal marker D21S1446.

[0143] This result indicates first of all that the telomeric DNAsequences have been entirely eliminated (both from maternal and fetalDNA). Secondly, the unclear signal of the DNA sequences of the proximalmarker D21S1411, is interpreted to be caused by a mixture of maternaland fetal DNA. Thirdly, we conclude that the three clear signals of thedistal marker D21S1446 (one of which is located in a position, which isnot the same as the maternal as seen in the electropherogram of the DNAfrom the lymphocytes of the maternal blood sample), are fetal, and showsthat the fetus has trisomy 21.

[0144] 9. Summary

[0145] This example illustrates that it is possible to routinelyenumerate numbers of chromosomes 21 and thus identify foetal trisomy 21Down syndrome by DNA analysis of a maternal blood sample, followingenzymatic digestion of telomeric and subtelomeric DNA sequences. Theprocess is rapid with results available within the same day of arrivalof the blood sample in the laboratory. Furthermore, substantialautomation is possible.

EXAMPLE 3B

[0146] 1. Pregnancy and Blood Sample

[0147] 12 ml of blood was drawn into 2 edetic acid (EDTA) tubes byvenipuncture from a pregnant woman at 12 weeks gestational age,following written informed consent with Ethical Approval from the LocalEthical Committee.

[0148] 2. Isolation of Cells from the Nuclear Compartment and Plasma

[0149] Both nucleated cells from the nuclear compartment and from plasmawere isolated from the maternal blood using a Triple Density Gradientaccording to the protocol as described (Ganshirt et al., DiagnosticCytogenetics, Springer Lab Manual, 1999 R. -D. Wagner, Fetal Cells inMaternal blood, pp 401-415) with slight modifications.

[0150] 12 ml of the EDTA blood is added to 12 ml of Phosphate BufferSolution (PBS) and mixed by inverting the tube. 6 ml of the blood/PBSmixture is pippetted into four 15 ml polystyrol tubes. Three layers ofPercoll^(R) (Amersham Pharmacia) are underlayered, using a long and thincanula attached to a syringe. Initially 3 ml of 40% Percoll isunderlayered, followed by 3 ml of 45% and 3 ml of 50% Percoll. Thesuspension is then centrifuged at 500 g for 30 min. The plasma layer andthe lymphocyte layer are removed and transferred to clean tubes andagain centrifuged at 500 g for 10 min. The cell pellet is washed in PBSand transferred to microtubes containing 5 μl proteinase K solution (400mg/l proteinase K, 20 mmol/l dithiothreitol, 1.7 μmol/l sodium dodecylsulfate, 10 mmol/l Trisbuffer, 50 mmol/l potassium chloride).

[0151] 3. Exonucleolytic Enzyme Digestion

[0152] 1 unit of Bal31 per microgram of DNA was used to yield a uniformladder of deletions (>1 kb) over a 20 minute period at 30° C. Thereaction was stopped by adding EDTA to a final concentration of 50 mM.

[0153] Enzymatic digestion is first performed on DNA of lymphocytes tocalibrate the concentration and time required for optimal elimination ofDNA sequences to allow differentiation between fetal and maternal DNA.The primers chosen in this case are D21S1446 and D21S1411 at 21q22.3(with D21S1411 being located proximal to D21S1446) together with theprimer for the telomere DNA sequence TTAGGG.

[0154] The optimal conditions to discriminate between maternal and fetalsignals was set for elimination of telomeres plus the marker D21S1446 inDNA of lymphocytes of the maternal blood sample (primarily maternalcells). This was evaluated as described in steps 4 and 5. The enzymaticdigestion of cells isolated from maternal plasma (a mixture of maternalcells with an enrichment of fetal cells) was then exposed to Bal31 underthese verified optimal conditions.

[0155] 4. Multiplex PCR

[0156] A multiplex fluorescence PCR assay is applied by routinetechnology (see e.g. Pertl et al., 1997 Am. J. Obstet. Gynecol. 177,899-906) using primers for D21S1411 and D21S1446. Each of the forwardprimers was labelled (5′ end) with a fluorescent dye to enablevisualisation and analysis of the PCR product. After the initialdenaturation, 24 cycles of 95° C. for 48 sec, 60° C. for 48 sec, and 72°C. for 1 min was performed followed by an extension of 72° C. for 15min.

[0157] 5. Analysis

[0158] The allelic fragments were resolved on a 6% denaturingpolyacrylamide gel, and analysed on an Applied Biosystems (Warrington)373 DNA sequencer running Genescan 672 software. Amplification productswere sized, and their fluorescence intensities were calculated, based onthe areas of the peaks seen on the electropherograms. Analysis wasperformed on (a) DNA from lymphocytes of the maternal blood sampledirectly, (b) DNA from same type of cells after enzymatic digestion, and(c) DNA from cells in the plasma component of the maternal sample(expected to contain an enrichment of fetal cells) after enzymaticdigestion.

[0159] 6. Results

[0160] (a) The electropherogram obtained from PCR products of DNA of thelymphocytes of the maternal blood sample shows the occurrence of twoequal sized peaks for each of the two primers D21S1411 and D21S1446.This result is the expected from a normal female.

[0161] (b) The electropherogram obtained from PCR products of DNA of thelymphocytes from the maternal blood sample, following enzymaticexonucleolytic digestion with 2 units of Bal31 for 20 min shows absenceof signals for the most distal marker D21S1446, while two signals areseen at positions for the proximal marker D21S1411.

[0162] This indicates that these conditions for enzymatic digestion areoptimal for our purposes of differentiating between maternal and fetalDNA.

[0163] (c) The electropherogram obtained from the DNA of cells inmaternal plasma, following enzymatic digestion under same conditions asin (b) shows unclear signals for the proximal marker D21S1411 but threeclear signals for the distal marker D21S1446.

[0164] This result indicates first of all that the telomeric DNAsequences have been entirely eliminated (both from maternal and fetalDNA). Secondly, the unclear signal of the DNA sequences of the proximalmarker D21S1411, is interpreted to be caused by a mixture of maternaland fetal DNA. Thirdly, we conclude that the three clear signals of thedistal marker D21S1446 (one of which is located in a position, which isnot the same as the maternal as seen in the electropherogram of the DNAfrom the lymphocytes of the maternal blood sample), are fetal, and showsthat the fetus has Trisomy 21.

SUMMARY

[0165] This example illustrates that it is possible to routinelyenumerate numbers of chromosomes 21 and thus identify foetal trisomy 21Down syndrome by DNA analysis of a maternal blood sample, followingenzymatic digestion of telomeric and subtelomeric DNA sequences. In thisexamples DNA is less degraded, and it has therefore been possible toperform the diagnostic analysis without the application of an adaptor.As with Example 3A analysis is rapid with results available within thesame day of arrival of the blood sample in the laboratory; andsubstantial automation is possible.

1. A method for the identification of foetal DNA in a maternalDNA-containing sample, said method comprising (a) isolating DNA fromsaid sample, (b) subjecting said DNA to exonucleolytic digestion by anenzyme so as to remove end regions of said DNA, and (c) detecting thepresence of a DNA sequence remaining in foetal DNA but absent frommaternal DNA as a result of said digestion process.
 2. A methodaccording to claim 1 wherein said maternal DNA-containing sample is ablood or vaginal sample.
 3. A method according to claim 1 of claim 2wherein in a preliminary step, cells are first isolated from maternalplasma, and DNA then isolated from these in step (a).
 4. A methodaccording to any one of the preceding claims wherein DNA from step (a)is cut using a restriction enzyme into fragments prior to step (b).
 5. Amethod according to claim 4 wherein cut ends of the DNA fragments areprotected prior to the exonucleolytic digestion so that they are notsusceptible to digestion by the exonucleolytic enzyme.
 6. A methodaccording to claim 5 wherein the restriction enzyme used to form the DNAfragments is one which cuts such as to provide binding sites for aprotecting moiety, and protection is achieved by ligation of thatprotecting moiety.
 7. A method according to claim 6 wherein theprotecting moiety is an adaptor.
 8. A method according to claim 7wherein the adaptor is 2′O-methyl ribonucleotide DNA or a complementaryoligonucleotide containing phosphorothioate links.
 9. A method accordingto any one of the preceding claims wherein foetal DNA is isolated frommaternal DNA.
 10. A method according to claim 9 wherein foetal DNAfragments are separated by hybridising biotinylated probes specific forteleomere DNA sequences remaining in foetal DNA after digestion, to saidDNA and thereafter immobilising said probes to a streptavidin coveredsupport.
 11. A method according to claim 10 wherein said strepavidincovered support comprises a paramagnetic particle.
 12. A methodaccording to any one of the preceding claims wherein the said DNAsequence remaining in foetal DNA is a telomere sequence or a chromosomemarker located proximal the telomere region.
 13. A method according toclaim 12 wherein said DNA sequence remaining in foetal DNA is a telomeresequence.
 14. A method according to claim 12 wherein said DNA sequenceremaining in foetal DNA is a sub-telomeric polymorphicchromosome-specific marker.
 15. A method according to any one of thepreceding claims wherein the said DNA sequence is detected using a firstlabelled probe which is specific for said sequence.
 16. A methodaccording to claim 15 wherein said label is a fluorescent label.
 17. Amethod according to any one of the preceding claims wherein theexonucleolytic enzyme is Bal31.
 18. A method according to any one ofclaims 1 to 16 wherein exonucleolytic digestion is effected in stages inwhich, in a first step, 3′ extension DNA is removed, in a second step,3′-5′ ss regions are excised and in a third step, ss regions aredigested.
 19. A method according to claim 18 wherein the first step iseffected using Mungbean nuclease.
 20. A method according to claim 18 orclaim 19 wherein the second step is effected using Exonuclease III. 21.A method according to any one of claims 18 to 20 wherein the third stepis effected using Mungbean nuclease.
 22. A method according to any oneof the preceding claims wherein the amount of DNA of foetal origin inthe sample is determined.
 23. A method according to any one of thepreceding claims wherein foetal DNA in the sample is amplified.
 24. Amethod according to claim 23 wherein the amplification is effected usingthe polymerase chain reaction (PCR).
 25. A method according to claim 24wherein PCR is used to quantitate the amount of foetal DNA in thesample, or the amplified sample is subjected to genetic analysis.
 26. Amethod according to any one of the preceding claims wherein foetal DNAidentified is subjected to pre-natal diagnosis.
 27. A method accordingto claim 26 wherein the diagnosis detects chromosome aberrations.
 28. Amethod according to any one of the preceding claims wherein before step(c), DNA is separated according to size.
 29. A method according to anyone of claims 25 to 27 wherein separated DNA identified as being offoetal origin is amplified with a labelled primer which is specific fora particular chromosome or diagnostic region of DNA.
 30. A methodaccording to claim 29 wherein said second primer carries a fluorescentlabel.
 31. A method according to claim 25 or claim 26 wherein foetal DNAis amplified using a fluorescently labelled primer, and the amplifiedsequence is diagnostic for a chromosome or DNA condition.
 32. A methodaccording to claim 31 wherein said primer amplifies a sequence which isspecific for chromosome 18, 21, 13, X or Y.
 33. A method according toclaim 25 which is used in the diagnosis of conditions of the mother. 34.A method according to claim 33 wherein the conditions are pre-eclampsia,in predicting the risk of pre-term labour, and in the later developmentof autoimmune disease.
 35. A kit for identifying foetal DNA in amaternal blood or vaginal sample, said kit comprising means forextracting DNA from a blood or vaginal sample, an exonucleolytic enzymecapable of digesting DNA, and a labelled primer suitable for detecting aspecific DNA sequence found in a terminal region of DNA.
 36. A kitaccording to claim 35 which further comprises a restriction enzyme whichcuts DNA so as to produce a binding site on the DNA for an adaptorsuitable for protecting the cut ends of DNA fragments from enzymaticdigestion.
 37. A kit according to claim 34 or claim 35 which furthercomprises an adaptor suitable for protecting the ends of DNA fragmentsfrom enzymatic digestion.
 38. A kit according to any one of claims 34 to37 which further comprises a biotinylated telomeric probe.
 39. A kitaccording to claim 38 which further comprises streptavidin coatedparamagnetic particles.
 40. A kit according to any one of claims 34 to39 which comprises a labelled primer which is diagnostic of a chromosomecondition.
 41. A method for the identification of foetal DNA in amaternal DNA-containing sample, substantially as hereinbefore described.42. A kit for the identification of foetal DNA in a maternalDNA-containing sample substantially as hereinbefore described.